PROJECT SUMMARY/ABSTRACT: Biomolecular condensates are dynamic, membraneless compartments that spatiotemporally regulate a myriad of cellular functions from gene transcription to cellular stress response. Liquid-liquid phase separation (LLPS) is increasingly appreciated as the biophysical mechanism for how these condensates assemble. Key to proper condensate function is the maintenance of their dynamics and assembly/disassembly processes, but little is known about these mechanisms. Hints are provided from disease states whereby condensates may undergo liquid-to-solid transitions into cytoplasmic inclusions that contain protein quality control components and are characteristic of proteinopathies such as amyotrophic lateral sclerosis. We have identified UBQLN2, a member of ubiquitin-mediated protein quality control systems, as a contributor to condensate function. We recently showed that UBQLN2 forms condensates in vitro, and is recruited to stress granules, cytoplasmic condensates that form in response to stress. The multitude of UBQLN2 functions are driven through interactions with proteasomal subunits, polyubiquitin chains, and client proteins. Ubiquitin and polyubiquitin, biological signals for the maintenance of protein homeostasis through degradation and autophagy, drive disassembly of UBQLN2 condensates in vitro. These observations have broad implications for how phase separation mechanisms regulate the function of protein quality control systems. In this project, we aim to identify the molecular and cellular mechanisms that drive how UBQLN2 condensates assemble and disassemble. Aim 1 determines how domain-domain interactions promote or inhibit phase separation of UBQLN2 via construction of phase diagrams for constructs from a combination of UBQLN2 domain deletion and disease-linked mutations. These domain deletions will be used to mimic the different ?states? of UBQLN2 when specific domains are engaged with binding partners and unable to contribute to LLPS. We will use UBQLN2 disease-linked mutations as a nature-provided library to elucidate how intra- and intermolecular UBQLN2 interactions promote or inhibit condensate assembly and alter condensate morphology and material properties both in vitro and in mammalian cell culture models. Aim 2 quantifies how UBQLN2 condensates are affected by UBQLN2 engagement with protein quality control components, including proteasomal receptors, client proteins, and different types of polyubiquitin chains. We monitor these effects in vitro and with designed mutants in vivo. Importantly, we develop a reconstituted UBQLN2 condensate model to quantify the parameters of how polyubiquitin and polyubiquitinated substrates engage with UBQLN2 to disassemble condensates. These studies will lay the foundation for determining the physiological roles of phase separation as it pertains to protein homeostasis through ubiquitin- mediated pathways.